Methods and compositions for inhibiting sterol biosynthesis



United States Patent Pennsylvania N0 Drawing. Filed Dec. 7, 1962, Ser. No. 242,932 5 Claims. (Cl. 167-65) Our invention is concerned with methods and compositions for reducing the levels of certain sterol materials from the plasma and organs of animal organisms.

Among the etiological factors for arteriosclerosis is an abnormally-high level of various steroidal lipids, notably cholesterol, in the plasma and organs of the particular subject and it has accordingly been an object of metabolic research to discover agents which would'inhibit the biosynthesis of cholesterol. To a large extent such an objective has been realized with several chemical agents which successfully prevent the formation of cholesterol. It appears however that mere inhibition of cholesterol synthesis not infrequently results in a concomitant and unduly large accumulation in the body of the cholesterol precursor sterols, such as lanosterol, zymosterol and particularly desmosterol.

We have now discovered a method for inhibiting biosynt-hesis of squ-a'lene. Since this substance serves directliy as a precursor in animal organisms for lanosterol, and therefore demosterol and cholesterol, the objective of in hibiting cholesterol biosynthesis without substantial accumulation of squalene, lanosterol, zymosterol and/or desmosterol is thus realized. In short, by virtue of inhibiting the synthesis at an early stage, the precursor thereby not utilized in cholesterol synthesis can be readily utilized by the body in other metabolic pathways. Since the site of inhibition is prior to the synthesis of any steroidal lipid, e.g., as squ'alene biosynthesis, the desired result of sterol inhibition is effected without such side effects as fatty infiltration, liver damage, hypertrophy of the adrenals and the like.

Our method involves the administration of a compound of the formula:

(IJa u lower alkyl lower alkyl advantageously be employed on a bid or t.i.d. basis. quite surprisingly not only do such compositions reduce lipid biosynthesis but in addition they appear to actually reduce absorption of ingested cholesterol from the gastrointestinal tract.

The phara'maceutical compositions of this invent-ion thus embrace quaternary salts of the above formula. The anionic halide moiety is of relatively minor importance since the properties of the compounds result from the cationic form of the steroidal compound. Thus the anion need only be a pharmaceutically acceptable nontoxic anion and may thus be for example iodide, chloride, bromide and the like. In fact, the highly singular properties of these compounds appear to be a function of the structure found in the A and B rings. Thus while the cholestane side chain at C-17 is preferred for economic reasons, other compounds containing the various pregnane or testosterone C-17 groups also demonstrate the properties herein described.

The preparation of the compounds utilized in this invention is more fully described hereafter. It involves the preparation of a 4-aza-5a-cholestane by treatment of a 3,5-seco-4-norcholestan-5-one-3-oic acid with a lower alkyl amine and subsequent hydrogenation. Introduction of the 3-benzyl group is next accomplished by treatment with benzylmagne-sium bromide followed by hydrogenation. Formation of the quaternary salt is then eifected by standard procedures, such as treatment with a lower alkyl halide.

The following examples will further typify the nature of our invention but should not be construed as a limitation thereof.

Example 1 3,S-seco-4-norcholestan-5-on-3-oic acid (20 g., 0.05 mole) is dissolved in ml. of absolute alcohol which has been previously saturated with met-hyla-mine. The solution is heated in a pressure vessel at 150 for five hours and then concentrated to about 50 ml. and allowed to cool overnight. The crystals which form are collected to yield 4-methyl-4-aza-5-cholesten-3-one, M.P. 98100.

. Recrystallization from ethanol produces white needles,

A solution of 10.5 g. of 4-methyl-4-aza-5-cholesten- 3-one in 150 ml. of glacial acetic acid is hydrogenated for 8 hours at 60 and 60 lb./in. pressure in the presence of 100 mg. of platinum catalyst. The solution is then filtered and the solvent distilled. The residue is dissolved in ether and washed with sodium bicarbonate solution and water. After drying the solution over anhydrous sodium sulfate, the ether is removed and the residue crystallized from aqueous alcohol. There is thus obtained 4-methyl-4-aza-5a-cholestan-3-one, M.P. 118 120". The melting point is raised at l21122 by further crystallization.

To a Grignard reagent prepared from 960 mg. (0.04 mole) of magnesium, 20 ml. of dry ether, and 3.8 g. (0.03 mole) of benzyl chloride is added under nitrogen with stirring, 4 g. (0.01 mole) of 4-methyl-4-aza-5acholestan-3-one in 20 ml. of toluene. The ether is removed by warming and an additional 20 ml. of toluene are added. The mixture is refluxed 12 hours and a saturated aqueous solution of ammonium chloride is then added at 0. The organic layer is separated and the aqueous layer extracted with ether. The combined organic extracts are washed with sodium chloride solution and dried over anhydrous sulfate. Removal of the solvent yields a a reddish oil which is very sensitive to air oxidation.

Without .further purification, 4.5 g. of the oil is dissolved in 150 ml. of 95% ethanol and one drop of 10% hydrochloric acid is added. The mixture is treated with hydrogen for 8 hours at 60 and 200 lb./in. pressure in the presence of 500 mg. of platinum catalyst. The catalyst is removed by filtration and the solution allowed to cool overnight. The resulting precipitate is collected and recrystallized from ethyl acetate to yield 3-benzyl-4-methyl- 4-aza-5a-cholestane as white needles, M.P. 122-l23. Two grams of 3-benzyl-4-methyl-4-aza-5a-cholestane is then treated with an excess of methyl iodide in ether. Isolation of the solid and washing with ether then yields the quaternary salt, 3-benzyl-4-methyl-4-aza-5u-cholestane I rnethiodide, M.P. 259260.

Alternatively by using other lower alkyl halides, as'for example ethyl chloride, the corresponding quaternary salts. are obtained.

Similarly methylamine may alkyl amine-s in the initial step of this procedure.

ExampleZ Ingredients: Amount, mg.

3-benzyl-4-methyl-4-aza-5wcholestane rnethiodide 10' Calcium sulfate, dihydrate 200 Sucrose 3'5 Starch 18 Talc 9 Stearic acid 3 The sucrose and calicum sulfate are thoroughly mixed and passed through a #40 mesh screen. Granulation is then effected with a hot 10% gelatin solution andthe wet granulation passed through a #4 mesh screen and dried for three hours at 120 F. The dried granulation is passed through a #14 mesh screen and mixed with the starch,

talc, stearic acid and 3-benzyl-4-methyl-4-aza-5ot-cholestane rnethiodide (which have previously been passed through a #60 mesh screen). The granulation is'then compressed into tablets employing a flat faced bevel edge single score punch and die.

One tablet is administered theree times a day.

. Example 3 Ingredients: Amount, mg.

3-benzyl-4-ethy1-4-aza-Sa-cholestane ethochloride 100 Magnesium stearate l Lactose 400 The above ingredients are passed through a #40 mesh screen, mixed well and introduced into a #40 hard gelatin capsule.

The above ingredients are mixed and introduced into a #1 hard gelatin capsule.

One capsule is administered two times a day.

What is claimed is:

1. The method of reducing stero i y he i n be replaced by other lower animal which comprises administering orally to said mal a compound of the formula:

C H11 CH l Q'CH'Q; V

lower alkyl' lower alkyl wherein X is a pharmaceutically acceptableiwnontoxic halide anion.

2.1The method :of reducing sterol'biosynthesis in an animal which comprises administering orally a pharmaceutical composition comprising a compound of the formula:

C )5 'i 33.3 Xe

lower alkyl lower alkyl CHs' Cs n CH3 hypocholester- References Cited by the Examiner Doorenbos: Drug Trade News, Sept. 3, 1962, pp. 48,

Kenna: I Chem. Soc. March 1960, pp. 945-952.

IULIAN'S. LEVITT,Primary Examiner.

FRANK CACCIAPAGLIA, JRL, LEWIS GOTTS,

. Examiners.

from about 10 mgpto about 

1. THE METHOD OF REDUCING STEROL BIOSYNTHESIS IN AN ANIMAL WHICH COMPRISES ADMINISTERING ORALLY TO SAID ANIMAL A COMPOUND OF THE FORMULA: 